PROJECT SUMMARY Pluripotent stem cells are essential during early embryonic development, and hold great potential for therapeutic applications. In both cases, correct regulation of division and differentiation of these cells is crucial for a favorable outcome. Currently, there are major gaps in our understanding of how stem cells maintain their pluripotent state, or make the transition to differentiation. Several types of non-coding RNA are enriched in stem cells, and have been proposed to function in these regulatory processes, in particular by affecting the chromatin state of the cells. We will leverage a prolific population of adult pluripotent stem cells, the neoblasts of the planarian Schmidtea mediterranea, to address the role and mechanism of two types of non-coding RNA in in vivo stem cell biology. One branch of this project concerns the role of long non-coding RNAs (lncRNAs) in pluripotency and differentiation. Whereas many in vitro screens have suggested a role for lncRNAs in the regulation surrounding pluripotency, few cases of in vivo effects have been demonstrated. We will use an in vivo screen of planarian stem cell function to first identify functionally important lncRNAs, which will then be studied further mechanistically. This will allow us to focus our efforts on the most relevant examples of lncRNA- mediated stem cell regulation. The other branch of this project focuses on the role of PIWI-interacting RNAs (piRNAs), and their binding partners the PIWI proteins, in stem cell biology. These small RNAs are mostly known for their role in transposon silencing in the animal germline, but are also essential for stem cell function, especially in regenerating animals. We aim to determine what the basis for that requirement is, by determining which stages of stem cell biology are affected and what the consequences of failure of the piRNA pathway are. Furthermore several aspects of piRNA function are not easily addressed in germ cells, and we will use the planarian stem cells to further our understanding of those mechanistic aspects of piRNA biology. This project will provide new insights in non-coding RNA function and mechanism, and will have broad impact on our understanding of pluripotent stem cells and regulation of genomic stability in general. These insights are essential for the safe use of pluripotent cells in therapeutic applications, as well as for targeted treatment of conditions related dysfunction of endogenous stem cells including forms of infertility, birth defects, and aging. Furthermore, this project has relevance to conditions related to loss of cell identity, such as cancer.